Synthesis and evaluation of novel D-ring substituted steroidal pyrazolines as potential anti-inflammatory agents

Abstract

To identify new potential anti-inflammatory agents, a number of novel steroidal derivatives with nitrogen heterocyclic side chains 4a-4l were synthesized and evaluated for their anti-inflammatory effects in activated RAW 264.7 macrophage cells. The synthesis scheme involves two steps, Claisen-Schmidt condensation with the corresponding pregnenolone and aromatic aldehydes as the first step followed by nucleophilic addition of thiosemicarbazide across an α, β-unsaturated carbonyl as a later step. Compound structures were confirmed by ¹H NMR, ¹³C NMR, HRMS, and IR. The compounds were assayed to test their anti-inflammatory effects in activated RAW 264.7 cells. Compound 4g, 3β-hydroxy-pregn-5-en-17β-yl-5'-(m-fluorophenyl)-4', 5'-dihydro-1'-carbothioic acid amido pyrazole, was identified as the most potent anti-inflammatory agent of the analysed compounds, with an IC₅₀ value of 0.86 µM on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells for 24 h compared to dexamethasone (IC₅₀ = 0.62 µM) and low cytotoxicity against RAW 264.7 cells. Compound 4g significantly inhibited NO produced by LPS-induced RAW 264.7 cells. Further studies showed that compound 4g markedly inhibited the expression of pro-inflammatory factors, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 cells. These results indicate that derivatives bearing pyrazoline structure might be considered for further research and scaffold optimization in designing anti-inflammatory drugs and compound 4g might be a promising therapeutic anti-inflammatory drug candidate.

Keywords: Anti-inflammatory activity; Pregnenolone; Pyrazoline; RAW 264.7 cells.